In ion analyzing equipment such as a mass analyzer, it is necessary to ionize the sample that is to be analyzed. Accordingly, an ionization apparatus is provided as a preliminary stage of these ion analyzers.
The electrospray ionization (ESI) method is available as one ionization method used in an ion analyzer.
The electrospray ionization (ESI) method is in wide use as one method that has the ability to ionize biological macromolecules such as DNA and proteins. With the ESI method, DNA and proteins can be ionized gently and complexes comprising multiple DNA or proteins formed by non-covalent bonding also can be ionized as is. In view of this feature, the ESI method is being used in structural functional analysis of biological molecules.
Biological activity is governed by the interactions of a variety of biological molecules. Accordingly, although the overall molecular mass of a complex formed by the interaction of biological molecules is important information in terms of understanding biological phenomena, ascertaining the strength of this interaction also is vital.
In order to analyze by mass analysis the strength of interaction between molecules of complex ions produced by the ESI method, use generally is made of a method that combines the ESI method with collision-induced activation. This method is as follows: A complex comprising a plurality of biological molecule subunits is ionized by the ESI method and molecular-weight-related ions of the complex are observed. Next, the ions are introduced into a vacuum, the ions are accelerated by an electric field and made to collide with gas molecules in vacuo to bring about collision-induced activity, the complex is dissociated into each of the constituents that form the complex and the correlation between the imparted collision activation energy and binding strength is investigated.
In the case of a complex of a protein-low-molecular weight compound, such as an enzyme and coenzyme or a drug acceptor and drug, this method is such that only the non-covalent bonds are severed and the individual molecules are dissociated without causing dissociation of the protein and low-molecular weight compound, allowing the strength of the interaction to be studied.
However, in a case where electrostatic-interactions and hydrogen bonding are the prime causes of formation of the complex and the structure of the constituent molecules is not sufficiently stable, as in the case of a complex comprising double-stranded DNA and protein, there are instances where not only the non-covalent bonds but also weak covalent bonds are broken and measured together with these fragments if the above-described method is applied. For example, severance of the covalent bonds of the double-stranded DNA portion of the complex also occurs. The reason for this is that the collision activation energy is consumed by both the dissociation and fragmentation of the complex. Consequently, the spectra obtained are complicated and it is difficult to quantitatively analyze the strength of the interaction accurately using the ordinary ESI—collision activation method. For example, refer to the following literature:
Activation Energies for Dissociation of Double Strand Oligonucleotide Anions: Evidence of Watson-Crick Base Paring in Vacuo, Schnier P D, Klassen J S, Strittmatter E F and Williams E R JACS (1998) 120, 9605-9613.
One other problem in the ion analysis of biological macromolecules is that there are cases where the solution of the biological sample unavoidably contains a surface-active agent. For example, a surface-active agent is necessary in order to extract liposoluble protein. If the solution of the biological sample contains a surface-active agent, it will be difficult to vaporize and ionize the protein with the ESI method.